Characterization of the cadmium complex of peptide 49-61: a putative nucleation center for cadmium-induced folding in rabbit liver metallothionein IIA

摘要

The synthetic peptide fragment containing residues 49-61 of rabbit liver metallothionein II (MT-II) (Ac-Ile-Cys-Lys-Gly-Ala-Ser-Asp-Lys-Cys-Ser-Cys-Cys-Ala-COOH), which includes the only sequential four cysteines bound to the same metal ion in Cd_7MT, forms a stable, monomeric Cd-peptide complex with 1:1 stoichiometry (Cd:peptide) via Cd-thiolate interactions. This represents the first synthesis of a single metal-binding site of MT independent of the domains. The ~(111)Cd NMR chemical shift at 716 ppm indicates that the ~(111)Cd~(2+) in the metal site is terminally coordinated to four side-chain thiolates of the cysteine residues. The pH of half dissociation for this Cd-peptide derivative, approx 3.3, demonstrates an affinity similar to that for Cd_7MT. Molecular mechanics calculations show that the thermodynamically most stable folding for this isolated Cd~(2+) center has the same counterclockwise chirality (#LAMBDA# or S) observed in the native holo-protein. These properties are consistent with its proposed role as a nucleation center for cadmium-induced protein folding. However, the kinetic reactivity of the CdS_4 structure toward 5, 5'-dithiobis (5-nitrobenzoate) (DTNB) and EDTA is greatly increased compared to the complete cluster (#alpha#-domain or holo-protein). The rate law for the reaction with DTNB is rate=(k_uf + k_(1, f) + k_(2, f) [DTNB]) [peptide], where k_uf=0.15 S~(-1), k_(1, f)=2.59x10~(-3) s~(-1), and k_(2, f)=0.88 M~(-1) s~(-1). The ultrafast step (uf), observable only by stopped-flow measurement, is unprecedented for mammalian (M_7MT) and crustacean (M_6MT) holo-proteins or the isolated domains. The accommodation of other metal ions by the peptide indicates a rich coordination chemistry, including stoichiometries of M-peptide for Hg~(2+), Cd~(2+), and Zn~(2+), M_2-peptide for Hg~(2+) and Au~+, and (Et_3PAu)_2-peptide.